1-prolyl-1-cysteinyl-1-tyrosine



Patented Aug. 7, T951 2,563,743 l-PROLYL-J-CYSTEINYL-l-TYROSINE' Albert Adolphe Plentl, New York, N. Y., assignor to Ralph L. Evans, Bay Shore, N. Y.

No Drawing. Application November 7, 1947, Serial No. 784,765

1 Claim. 1

.The present invention relates to starting nuclei for the preparation of polypeptide chains and. in particular to new and useful peptides resulting from the linkage of proline and cysteine.

Objects and advantages of the invention will be set forth in part hereinafter and in part will be obvious herefrom, or may be learned by practice with the invention, the same being realized and attained by means of the processes, steps, and combinations pointed out in the appended claim.

The invention consists in the novel steps, processes, combinations and, improvements herein shown and described.

From the Work which I have done it appears that proline, cysteine and tyrosine are some of the essential amino acids in certain physiologically active peptides secreted by the posterior lobe of the pituitary gland.

It is an object of'the'present invention to provide starting nuclei for building of polypeptide chains, said nuclei being the free dipeptide prolylcysteine, and also forms of this dipeptide in which one or more of the reactive groups not in peptide linkage are blocked with easily removable radicals. A further object is to provide similar starting nuclei in which a blocked or unblocked tyrosine radical is attached to the cysteine portion of the chain.

An example of preparation of embodiments of this invention is as follows:

Carbobenzoazy-l -proZyZ- (S -benz1/l) -1 cysteinc ethyl ester 40 g. of S-benzylcysteine ethyl ester HCl are dissolved in 200 cc. water cooled in ice, and sufiicient 20% NazCOa added to liberate the free base. The product separates as a heavy oil which is extracted with 3 X 150 cc. portions of ethyl acetate. The combined ethyl acetate extracts are dried with anhydrous MgSO i, filtered through folded filter paper and the filtrate concentrated to about 5% HCl and the ether-ethyl acetate layer sepa- Ca-IC.: C, 63.83 H, 6.38 N, 5.96 S, 6.81

Found: 63. 74 6. 34 6. 6. 59 63. 76 6. 28 6. 05 6; 51

Carbobenzozry-I -proZyZ-S-benzyl-1-cysteine hydrazide 12.0 g. of carbobenzoxy prolyl-S-(benzyD-cysteineethyl ester are dissolved in 200 cc. of absolute ethyl alcohol. Slight warming may be necessary to bring all of it into solution. To the clear solution are added cc. of hydrazine hydrate and the mixture is allowed to stand in the ice- At the end of this time the box for 72 hours. product begins to crystallize, and after an addi tional 24 hours, most of the product will have separated. The crystals are filtered off and recrystallized from dilute ethyl alcohol M. P. 139- 140. 4 or 5 recrystallizations may be necessary. obtained.

before the correct melting point can be 8.2 g. of the pure product are obtained.

C zaHza O lNlS (455) Galc: N, 12. 28%

Found: 12. 51 12. 25

Carbobenzowy-I -prolyl-1 S-benzyl) -1 -cysteinyl- 1 -tyrosine ethyl ester 8.0 g. of carbobenzoxy prolyl-S-benzyl-cysteinyl hydrazide are dissolved in 50 cc. acetic acid containing about glacial acetic acid. The solution is filtered through coarse paper and the filtrate cooled in ice. To this solution are added 1.20 g. (calculated amount) of sodium nitrite dissolved in about 3 cc. of water. The azide precipitates as an oil and is extracted with cold ether. A compound of unidentified nature precipitates between the ether and the water layer and is removed by filtration. The ethereal solution of the azide is dried with magnesium sulfate and after removal of the drying agent, this solution is added to 3.63 g. of tyrosine ethyl ester M. P. 105 C. dissolved in cc. anhydrous ethyl acetate. After standing at room temperature for 24 hours the crystalline product which had formed is removed by filtration and dried in open air. 4.40 g. of the crude product is obtained. The compound can be recrystallized from ether-ethyl acetate or acetone-water mixtures. From the latter solvent mixture the compound precipitates in gelatinous form and is difiicult to recrystallize, but the purification is much more efficient than from etherethyl acetate mixtures. The pure compound melts at 155156 C.

Calc.:C, 64.4 H, 6.15 N, 6.63 s, 5.06

Found: 64.2 6.57 6.95 4.89 63. 98 6. 41 7. 12 4. 79

Carbobcnzoxy prolyl- (S-benzyl) -1 -.cystei.nyZ-

. 1 -tyrsine 2.0 g. of the tripeptide ester M. 1 155-156 are finely ground in a mortar and shaken with an excess of 2N NaOH at room temperature until most of it had dissolved. The cloudy solution is extracted with ethyl acetate and the aqueous layer acidified with dilute hydrochloric or sulfuric acid. Thef-ree'acid precipitates as an oil which crystal- 112653111 the icebox after standing over night. After several recrystallizations from dilute ethyl alcoholthepure substance melts at 204 C. A total of-1.70 g.;is obtained.-

Cz'ilcultitedfor'fCyHisOiN3S Calm: o, 6355 H,"5.78 N,e.94 s, 5.28

Found: 63. 9 5. 98 7. i0 5. 07

M01. wt.

Ca1c.: 605

Found: 620, 595, 631; 618

1=Prblyl=1-cysteinyb1 -ty'rosz1ze- 40 cc. anhydrous liquid ammonia are collected in: a.200 cc: 3neck round bottom flask.- The flask iscooledin Dry-Ice-acetone mixture and provided with an eincient stirrer,'1.'20 g. of carbobenzoxy- 1 prolyl-l-(S-benzyl) -cysteinyl-tyrosine is dissolved in the ammonia'and 0.2 g; metallic sodiumis add'edinsmall amounts. When a permanent blue color is obtained, 500 mgm. of ammonium sulfate added. which decolorizes the solution almost immediately.

The stirrer is now removedand replaced by a gasi-nlet tube.- Nitrogen (O. P.) is blown through the'mixturein orderto avoid oxidation by atmospheric oxygen and to help =in the spontaneous evaporationorammonia. After 24 hours of this treatment a small amount of white -crystalline material remains in the fiask with almost no.residual ammonia. This residue is dissolved in. 30 cc. of water and kept under vacuum at room temperature. for.2 hoursand brought to pH 3-5 with N H2804.

A suspension of freshly prepared C1120 is then added in small amounts at a time. The suspension is stirred for 10 hours and then allowed to stand at room temperature for 24 hours. The amount of C1120 used is so adjusted that a slight excess is present at the end of the procedure. The precipitate is centrifuged and washed four times with 10 C0,; portionsrof water.

The Washed copper salts are suspended in cc. of water and hydrogen sulfide blown through this suspension for 45 minutes. The copper sulfides .are .thenremoved, preferably by filtration using filter cell, and the clear filtrate concentratedlunderreducedi pressure. The pH of this solution-is between 5 and 6. When the volume reaches less...than.1.0 cc. a fine crystalline precipitate appears which increases in amount upon standing in the icebox. This material is recrystallized from water from which it precipitates in form of fine needles. r The." substance is soluble. in: dilute acidzan alkali and precipitates. upon neutralization :of' its. acid.or'alkalinesolutions. Thernitroprussideandit Sullivanrreactions are: strongly. positivegswhich 'in.-;:.-

The .invention in..its broader aspects .is.. n'ot" limited. to thespecifi'cprocesses andjsteps. shown. and described but departures may be .made'theree fromwithin the scope .of theaccompanyingclaim 'without.departingjrom the principlesof the invention and. without sacrificing. its. chief 'advan-..

tage. v

What is claimed is: l-prolyl-lecysteinyleletyrosine.

ALBERT ADOLPHE PLENTL- REFERENCES CITEDI The-following references are of record: in. the: 

